MicroRNAs are predicted to regulate a majority of genes in human cells and both overexpression and loss of expression of some miRNAs are correlated with malignant phenotypes in different cancer cells. A decrease in miRNA activity is most commonly observed and may be important for the plasticity of tumor cells to undergo transitions between differentiation states and to grow in different niches. One of the major objectives of this proposal is to investigate the functions of subsets of miRNAs in contributing to the properties of tumor cells and specifically to the ability of these cells to respond to stress. Many therapeutic treatments of cancer induce stress due to DNA damage, presence of unfolded proteins, deprivation of survival signals, and oxygen. We found that loss of miRNA regulation renders many types of cancer cells hypersensitive to such stress agents. For example, deletion of Dicer, a double strand endonuclease required for synthesis of most miRNAs, in many cancer cells yields viable cells that can in fact produce tumors in xenograft challenges but are hypersensitive to stress. Dicer- null human cancers have not been observed but recently sequencing of a haploid Dicer gene in several different types of human cancers identified a specific point mutation that inactivated one RNase III domain. This indicates that these tumor cells are being strongly selected for loss of a subset of miRNAs and for retention of other miRNAs. Cells expressing this mutant Dicer are defective for expression of the let-7 miRNA family but express near normal levels of other miRNAs. The four specific aims of this proposal build from progress over the past four years. First, we will further investigate the nature of factors targeting Argonaute 2 (Ago2) to specific RNA sequences in Dicer null cells. We will also investigate how this targeting enhances the quantitative level of silencing by co-binding miRNA. Second, we will characterize the subset of miRNAs generated by mutations of specific functions of Dicer and determine the consequences of expression of these subsets on tumor phenotype and response to stress. Third we will test if an inhibitor of Dicer should be considered as a promising objective for drug development to treat cancer in conjunction with chemotherapy. And fourth, we will investigate the function of Argonaute that is essential for viability in embryonic stem cells defective for synthesis of miRNAs, i.e. null for Dicer!